JP2005516581A - Ex vivo priming to produce cytotoxic lymphocytes specific for non-tumor antigens to treat autoimmune and allergic diseases - Google Patents

Abstract

Cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derived from IgE molecules are stimulated in vitro by stimulating resting natural CD8 T cells with IgE peptides presented by artificial antigen-presenting cells. Can be generated. IgE-specific CTL lyses target cells loaded with IgE peptides in vitro and suppresses antigen-specific IgE responses in vivo. Furthermore, adoptive transfer of IgE-specific CTLs into an asthmatic mouse model can reduce the occurrence of lung inflammation and airway hypersensitivity. IgE-specific CTLs provide treatment for allergic asthma and other IgE-mediated allergic diseases. Antigenic peptides identified from non-tumor self-antigens induce specific cytotoxic T lymphocytes (CTL) in vitro. CTL induced by peptides identified from CD40L can kill activated CD4 T cells. CTL generated in vitro specific for CD40L suppresses CD4-dependent antibody responses of all isotypes in vivo. In contrast, CTLs induced by antigenic peptides derived from IgE specifically suppress IgE responses, and early adoptive transfer of CD40L-specific CTLs to NOD mice delays the onset of diabetes in NOD mice . In vitro generated CTLs specific for non-tumor self antigens expressed on activated CD4 T cells modulate the immune response in vivo.

Description

Relationship with Related Application This application claims the priority of Patent Document 1 filed on May 15, 2001, the contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION An immune response against foreign antigens such as found in bacteria and viruses protects and eliminates infection. However, an abnormal immune response can cause allergic and autoimmune diseases. Immune responses to foreign, sometimes innocuous substances such as pollen, dust mites, food antigens and bee needles can result in allergic diseases such as hay fever, asthma and systemic anaphylaxis. Immune responses against self antigens such as islet antigen and cartilage antigen can lead to diabetes and arthritis, respectively. A prominent feature of allergic disease is activation of CD4 T cells and high production of IgE by B cells, whereas prominent features of autoimmune disease are activation of CD4 T cells and overproduction of inflammatory cytokines. Current therapies have focused on the treatment of symptoms of allergies and autoimmune diseases and do not prevent the onset and progression of the disease.

  CTL recognizes antigenic peptides derived from resting natural CD8 T cells and presented by major histocompatibility complex (MHC) class I molecules. When resting CD8 T cells encounter an antigenic peptide / MHC complex presented by specialized presenting cells, CD8 T cells are activated and differentiate into armed CTLs. Upon recognition of the peptide / MHC complex on the target cell, the antigen-specific CTL delivers a lethal blow and lyses antigen-expressing cells such as target cells or tumor cells infected with the virus.

  Natural T cell activation in vivo is controlled by many receptor-ligand interactions between T cells and specialized APCs such as dendritic cells (1). It is generally accepted that natural T cell activation requires two signals (Non-Patent Document 2). Each signal 1 is induced by the interaction of TCR and MHC / peptide complex (Non-Patent Document 3), and the non-polymorphic region of MHC class II / I molecule of CD4 / CD8 co-receptors (co-receptors) (Non-patent Document 4). Signal 2 is qualitatively different from signal 1 and is delivered, for example, through the interaction of CD28 and B7 via a T cell costimulatory molecule that interacts with a complementary ligand on APC. 5; 6). Signals 1 and 2 function synergistically and induce a series of signaling reactions that ultimately induce T cells to proliferate, produce cytokines, and differentiate into effector cells (7). 8). However, the relationship between signals 1 and 2 is unknown.

Although various molecules have been reported to have a costimulatory function, special attention has focused on costimulation delivered via the CD28-B7 interaction (9). CD28 is a molecule containing a single Ig-like domain and is constitutively expressed as a homodimer on T cells (Non-Patent Document 5). CD28 molecules transmit unique signals that stimulate T cells to produce growth-promoting cytokines such as IL-2 through interaction with either B7-1 or B7-2 molecules on APCs (non-patented). Document 10), and up-regulate the expression of survival factors (survival factor), such as _{Bcl-X L} (non-patent document 11), and to prevent anergy induced by signal 1 alone (non-Patent Document 12) then It is considered.

  Another molecular pair that has an important role in T cell activation is LFA-1 / ICAM-1 (Non-patent Document 13). ICAM-1 belongs to the Ig gene superfamily and has 5 Ig C-like domains in the extracellular region; it is expressed in both hematopoietic and non-hematopoietic cells. The receptor for ICAM-1 on T cells is LFA-1 (CD11 / CD18), which belongs to the b2 integrin family (Non-Patent Document 14). The interaction between LFA-1 and ICAM-1 has a potent costimulatory function for T cells (Non-patent Document 15), but this function is reflected in another signal pathway, or T cells Opinion about whether it is reflected in the increase in adhesion between APC and APC is different (Non-Patent Document 16:17).

  In addition to the B7 and ICAM-1 molecules, several other molecules on APC, including CD70 (18) and thermo-stable antigen (HSA) (19), are associated with them on T cells. Can exert an extremely powerful costimulatory function through their interaction with their respective ligands. This means that T-APC interactions are highly complex and that many interactions are involved between complementary sets of T cells and molecules on APCs. The interaction of each set of molecules elicits a specific signal that induces a different cellular event. Different signal combinations can act synergistically on optimal T cell activation and determine the ultimate fate of the T cell. Alternatively, the function of the costimulatory molecules may overlap and the signal induced by each set of costimulatory molecules may be additive. The need for each set of costimulatory molecules will be influenced by the strength and properties of signal 1.

In view of these two possibilities, it is important to understand the minimum requirements for natural T cell stimulation. Experiments with CD28 ^{− / −} mice show that CD28-B7 interaction is highly important in some situations, but not in others (20). Similarly, the requirement for the LFA-1 / ICAM interaction of the primary reaction is not a constant finding (Non-patent Document 21).

  CD8 T cells recognize antigenic peptides derived primarily from viral proteins and proteins expressed on tumor cells. Recently, however, it was reported that a newly synthesized protein was preferentially processed by an antigen-processing mechanism (Non-patent Document 22). Upon activation, immune cells acquire the ability to synthesize a number of novel proteins, and IgE-producing B cells and activated CD4 T cells produce a different set of peptide / MHC complexes from non-IgE producing cells and resting CD4 T cells. It is possible to present. These peptide / MHC complexes presented on IgE-producing B cells and activated CD4 T cells can be recognized by CD8 T cells. Thus, CTL specific for these peptide / MHC complexes could treat allergies and autoimmune diseases. However, a number of immune tolerance mechanisms were able to prevent activation of CD8 T cells towards self antigens in vivo.

  CD8 lymphocytes (CTLs) are adoptive immunity weapons responsible for the recognition and elimination of infected, tumor and allogeneic cells. Once primed, CTLs recognize their target antigens on a wide variety of cells and lyse target cells and / or secrete cytokine-like TNF-alpha or IFN-gamma to function them. Can be fulfilled.

Presentation of antigen to CD8 ⁺ CTL (cytotoxic T lymphocyte) occurs in the context of MHC class I molecule (MHC-I), but presentation of antigen to CD4 ⁺ HTL (helper T lymphocyte) Occurs in the context of MHC class II molecules.

Efficient induction of CD4 ⁺ T cells requires that T cells interact with antigen presenting cells (APC), ie cells that express MHC class II and costimulatory molecules. APCs are dendritic cells, macrophages and activated B cells. Almost all nucleated cells express MHC-I, but natural CTL also requires presentation of antigen (Ag) by APC induced by bone marrow for efficient priming (Non-patent Document 23). . Dendritic cells are highly potent inducers of CTL responses (Non-Patent Document 24) and are thought to be the major APCs involved in CTL priming. Once primed, CTLs can recognize their cognate Ag on a wide variety of cells and respond by lysing target cells and / or secreting cytokines.

  Bone marrow-induced APC requires efficient CTL response to be primed (25), but activated CTLs immediately recognize and respond to Ag presented by a wide variety of cells. Can do. Induction of tumor- or virus-specific CTL immune responses in vivo has been shown to be dependent on bone marrow-derived antigen-presenting cells (Non-Patent Documents 26; 27). In general, bone marrow-derived APCs are accepted to take up cellular antigens either in the form of soluble antigens associated with chaperonin molecules or by phagocytosis through a unique mechanism for these cells.

Responses to cellular antigens have long been shown to be dependent on the assistance carried by CD4 ⁺ T cells. It has also been shown that cellular antigens must be presented on the same APC for recognition by CTL and HTL. The nature of this assistance has been interpreted as the need for IL-2, which is necessary for CTL expansion. Recent studies have shown that this assistance results from activation of dendritic cells by HTL and is mediated through CD40-CD40L interactions (28).

Thus, a similar scenario for the induction of a CD8-mediated immune response against cellular antigens (derived from tumor cells or infected cells) is as follows: Dendritic cells acquire antigens derived from tumors or infected cells. The interaction between DC-antigen and CD4 cells allows DC to activate CD8 cells.
US Provisional Patent Application No. 60 / 291,300 R. M.M. Steinman, Annu. Rev. Immunol. (1991) 9: 271-296 C. A. Janeway and Bottomry, Cell (1994) 76: 275-285. R. N. Germany, Cell (1994) 76: 287-299. M.M. C. Micheli and J.M. R. Parnes, Adv. Immunol. (1993) 53: 59-122 P. S. Linsley and J.M. A. Ledbetter, Annu. Rev. Immunol. (1993) 11: 191-212. Lenschow et al. , Annu. Rev. Immunol. (1996) 14: 233-258 Mueller et al. , Annu. Rev. Immunol. (1989) 7: 445-480 A. Weiss and D.C. R. Littman, Cell (1994) 76: 263-274 R. H. Schwartz, Cell (1992) 71: 1065-1068. June et al. , Immunol. Today (1994) 15: 321-331) Boise et al. , Immunity (1995) 3: 87-98. R. H. Schwartz, Curr. Opin. Immunol. (1997) 9: 351-357. Van Seventer et al. , J .; Immunol. (1990) 144: 4579-4586 T.A. A. Springer, Cell (1994) 76: 301-314 Shimizu et al. , Immunol. Rev. (1990) 114: 109-143 Damle et al. , J .; Immunol. (1993) 151: 2368-2379 Bachmann et al. , Immunity (1997) 7: 549-557. Hintzen et al. , J .; Immunol. (1995) 154: 2612-2623. Liu et al. , J .; Exp. Med. (1992) 175: 437-445. Shahinian et al. , Science (1993), 261: 609-612. Shier et al. , J .; Immunol. (1996) 157: 5375-5386 Schubert et al. , Nature, (2000) 404: 770-774. Dalyot-Herman et al. , J .; Immunol. 165 (12): 6731-6737 J. et al. Bancherean and R.M. M.M. Steinman, Nature, (1998) 392: 245-252) P. J. et al. Fink and M.M. J. et al. Bevan, Exp. Med (1978) 148: 755-766. Paglia et al. , J .; Exp. Med. (1996) 183 (1): 317-322 Labeur et al. , J .; Immunol. (1999) 162 (1): 168-175 S. R. Clarke, J .; Leukocyte Bio (2000) 67 (5): 607-614

SUMMARY OF THE INVENTION Immune cells such as IgE-producing B cells and activated CD4 T cells play a central role in the pathogenesis of allergic and autoimmune diseases. The present invention utilizes cytotoxic T lymphocytes (CTLs) to eliminate or suppress immune cells that cause allergies and / or autoimmune diseases. That is, the onset and progression of the disease is prevented or interrupted by the method of the present invention.

The present invention provides a method for producing CTLs specific for one or more non-tumor autoantigen T cell epitopes;
a) isolating CD8 ⁺ T cells from the individual;
b) adding T cell epitopes to antigen presenting cells (APC's) with class I MHC molecules;
c) culturing CD8 ⁺ T cells with APC's loaded with antigen for a time sufficient to activate precursor CD8 ⁺ T cells specific for T cell epitopes;
d) increase activated CD8 ⁺ T cells in the culture in the presence of components necessary for the proliferation of activated CD8 ⁺ T cells; and e) recover CD8 ⁺ T cells from the culture;
Comprising that.

The present invention also provides CD8 ⁺ T cells that are specifically cytotoxic to disease-causing target cells, where the target cells bind one or more non-tumor self-antigens T to which they bind class I MHC molecules. It has cellular epitopes and CD8 ⁺ T cells are already selectively activated by interaction with class I MHC molecules that bind to non-tumor autoantigen T cell epitopes.

The invention also provides a method for treating a disease mediated by a target cell that causes the disease, wherein the target cell has one or more non-tumor self-antigen T cell epitopes that bind to class I MHC molecules on its surface. The method comprises administering activated CD8 ⁺ T cells to a patient in need of such treatment, wherein the CD8 ⁺ T cells bind to non-tumor autoantigen T cell epitopes. Is already selectively activated by interaction with.

The present invention creates and uses artificial antigen-presenting cells to destroy CD8 T cell immune tolerance to self-antigens and CTLs specific for antigenic peptides identified from IgE or CD40L proteins Indicates that has occurred. Adoptive transfer of in vitro generated CTLs specific for CD40L to NOD mice dramatically delays the onset of diabetes, and CTLs specific for IgE peptides suppress IgE production and asthma Reduced lung inflammation in a mouse model. The above system is also potentially applicable to human diseases caused by CD4 T cells and IgE producing B cells. Autoimmune diseases caused by CD4 T cells are diabetes, rheumatoid arthritis, SLE, multiple sclerosis and psoriasis. On the other hand, allergic diseases mediated by IgE are systemic anaphylaxis, allergic rhinitis, food allergies and allergic asthma caused by drugs, poisons and peanuts. In addition, other self-antigens expressed on immune cells can be used to generate CTLs in vitro as well as in vivo in the treatment of autoimmune and allergic diseases. RNA and DNA encoding non-tumor antigens expressed on antigenic peptides, proteins or non-tumor cells can also be used to develop vaccines for treating or preventing allergies and autoimmune diseases.
Detailed Description of the Invention In one aspect, the present invention provides a method of treating an individual with a non-tumor autoantigen T cell epitope comprising:
a. A naturally occurring antigen presenting cell (APC) or a non-naturally occurring antigen presenting cell line (nnAPC) is prepared, wherein the APC or the nnAPC is up to about 15 associated with allergies and / or autoimmune diseases. Different peptide molecules, preferably about 10 different peptide-epitope molecules can be presented, at the same time each peptide is about 6-12 amino acids long, and preferably about 8-10 amino acids long And a concentration range of about 10 nM to 100 μM;
b. Recovering CD8 ⁺ cells from the individual or a suitable donor;
c. Stimulating the CD8 ⁺ cells with the APC or the nnAPC cell line;
d. Adding the CD8 ⁺ cells to a medium containing cytokines such as IL-2, IL-7 or CGM, preferably IL-2, or a combination of IL-2 and IL-7;
e. Mixing non-suspended peripheral blood monocytes or CD8-deficient peripheral blood monocytes recovered from the individual or a suitable donor with about 10-50 μg / ml peptide;
f. The peripheral blood monocyte suspension is in the range of about 3,000 to 7,000 rads, preferably 5,5, required to sterilize all components in the suspension except the desired peripheral blood monocytes. Irradiation with a sufficient dose of gamma radiation, such as 000 rads;
g. Isolating adherent peripheral blood monocytes;
h. About 10 ng / ml to 10 μg / ml of each peptide is loaded on the adherent peripheral blood monocytes;
i. Combining the CD8 ⁺ cells and the adherent peripheral blood monocytes at a ratio of about 10 CD8 ⁺ cells to 1 adherent peripheral blood monocytes;
j. Stimulating the suspension, optionally combined with CD8 ⁺ cells and peripheral blood monocytes, for about 6-7 days;
k. Optionally stimulating the suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in medium;
l. Optionally assaying CD8 ⁺ suspension for appropriate CTL activity and optionally assaying for CTL purity, sterility and endotoxin content; and m. Inoculating the individual with a CD8 ⁺ suspension;
Comprising that.

In another aspect, the invention provides a method of treating an individual, the method comprising:
a. A naturally occurring antigen presenting cell (APC) or a non-naturally occurring antigen presenting cell line (nnAPC) is prepared, wherein the APC or the nnAPC is up to about 15 associated with allergies and / or autoimmune diseases. Different peptide-epitope molecules, preferably about 10 peptides can be presented, at the same time each peptide is 8-10 amino acids long;
b. Collecting CD8 ⁺ cells from the individual;
c. Stimulating the CD8 ⁺ cells with the APC or the nnAPC cell line for about 6-7 days;
d. Stimulating the CD8 ⁺ cells with IL-2 and IL-7 in medium;
e. Peripheral blood monocytes collected from the individual are mixed with about 20 μg / ml of each peptide;
f. Irradiating the CD8-deficient peripheral blood monocyte suspension with about 5,000 rads of gamma radiation;
g. Isolating adherent peripheral blood monocytes;
h. Loading the adherent peripheral blood monocytes with about 100 ng / ml of the epitope;
i. Combining the CD8 ⁺ cells and the adherent peripheral blood monocytes at a ratio of about 10 CD8 ⁺ cells to 1 peripheral blood monocyte;
j. Stimulating the suspension of CD8 ⁺ cells and peripheral blood monocytes for about 6-7 days;
k. Stimulating the suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in medium;
l. Assay the CD8 ⁺ suspension for proper CTL activity, purity, sterility and endotoxin content; and m. Inoculating the individual with a CD8 ⁺ suspension;
Comprising that.

Other aspects of the invention include, but are not limited to, rheumatoid arthritis, lupus, psoriasis, autoimmune nephritis, multiple sclerosis, insulin-dependent diabetes, autoimmune thyroiditis, Crohn's disease, inflammatory bowel disease, transplantation Methods for treating individuals with autoimmune diseases including unilateral host disease and transplant rejection and / or allergic diseases including but not limited to food allergy, hay fever, allergic rhinitis, allergic asthma and snake venom allergy This method provides:
a. A naturally occurring antigen presenting cell (APC) or a non-naturally occurring antigen presenting cell line (nnAPC) is prepared, wherein the APC or the nnAPC is up to about 15 different peptides associated with such diseases- Epitope molecules, preferably about 10 peptides can be presented, at the same time each peptide is 8-10 amino acids long;
b. Collecting CD8 ⁺ cells from the individual;
c. Stimulating the CD8 ⁺ cells with the APC or the nnAPC cell line for about 6-7 days;
d. Stimulating the CD8 ⁺ cells with IL-2 and IL-7 in medium;
e. Peripheral blood monocytes collected from the individual are mixed with about 20 μg / ml of each peptide;
f. Irradiating the CD8-deficient peripheral blood monocyte suspension with about 5,000 rads of gamma radiation;
g. Isolating adherent peripheral blood monocytes;
h. Loading the adherent peripheral blood monocytes with about 100 ng / ml of the epitope;
i. Combining the CD8 ⁺ cells and the adherent peripheral blood monocytes at a ratio of about 10 CD8 ⁺ cells to 1 of the peripheral blood monocytes;
j. Stimulating the suspension of CD8 ⁺ cells and peripheral blood monocytes for about 6-7 days;
k. Stimulating the suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in medium;
l. Assay the CD8 ⁺ suspension for proper CTL activity, purity, sterility and endotoxin content; and m. Inoculating the individual with a CD8 ⁺ suspension;
Comprising that.

  Another aspect of the invention is a method for the treatment of allergies and / or autoimmune diseases, wherein nnAPC presents the following peptides, SEQ ID NO: 15 to SEQ ID NO: 49.

  Another aspect of the invention is a method of treating a non-cancerous disease or disease state that results in an inadequate or immature immune response, usually associated with class I HLA molecules, wherein the treatment is infected, Alternatively, transformed cells were eliminated and the exclusion was proven to be mediated by CTLs.

Another aspect of the invention is a method of treating a non-cancerous disease or disease state that results in an inadequate or immature immune response that is usually associated with class I HLA molecules, where it is susceptible to exclusion by CTLs. Infected or transformed cells that have been shown to be:
a. A naturally occurring antigen presenting cell (APC) or a non-naturally occurring antigen presenting cell line (nnAPC) is prepared, wherein the APC or the nnAPC is up to about 15 associated with such a disease or disease state. Different peptide molecules, preferably about 10 peptide epitope molecules can be presented, at the same time each peptide is 6-12 amino acids long, preferably 8-10 amino acids long, and about 10 nM-100 μM A range of concentrations;
b. Recovering CD8 ⁺ cells from the individual or a suitable donor;
c. Stimulating the CD8 ⁺ cells with the APC or the nnAPC cell line;
d. Adding the CD8 ⁺ cells to a medium containing cytokines such as IL-2, IL-7 or CGM, preferably IL-2, or a combination of IL-2 and IL-7;
e. Mixing non-suspended peripheral blood monocytes or CD8-deficient peripheral blood monocytes recovered from the individual or a suitable donor with about 10-50 μg / ml peptide;
f. The peripheral blood monocyte suspension is about 3,000-7,000 rads, preferably 5,000 rads, required to sterilize all components in the suspension except the desired peripheral blood monocytes. Irradiation with a sufficient dose of γ-radiation such as
g. Isolating adherent peripheral blood monocytes;
h. About 10 ng / ml to 10 μg / ml of each peptide is loaded on the adherent peripheral blood monocytes;
i. Combining the CD8 ⁺ cells and the adherent peripheral blood monocytes at a ratio of about 10 CD8 ⁺ cells to 1 peripheral blood monocyte;
j. Stimulating the suspension, optionally combined with CD8 ⁺ cells and peripheral blood monocytes, for about 6-7 days;
k. Optionally stimulating the suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in medium;
l. Optionally assaying the CD8 ⁺ suspension for appropriate CTL activity and optionally assaying for CTL purity, sterility and endotoxin content; and m. Inoculating the individual with a CD8 ⁺ suspension;
Is processed by a method comprising:

The present invention provides non-naturally occurring antigen-presenting cells (nnAPCs) derived from Drosophila melanogaster cells transfected with DNA for expression, where nnAPCs are allergic and / or autoimmune Up to 15 different peptide molecules related to disease can be presented simultaneously, preferably 10 peptide molecules encoded by DNA.

The present invention provides a non-naturally occurring antigen-presenting cell (nnAPC) derived from Drosophila melanogaster cells transfected for expression with human class I HLA, binding and co-stimulatory molecule DNA. Where nnAPC can simultaneously present up to 15 different peptide molecules, preferably 10 peptide molecules simultaneously encoded by DNA, which are associated with allergies and / or autoimmune diseases.

  Another aspect of the present invention provides nnAPCs that present peptides associated with various desired functions that enhance the treatment of individuals. For example, in addition to peptides associated with the disease or disease state being treated, nnAPC is a lymphocyte functional antigen (LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1 (ICAM-1), T-cells Peptides associated with accessory molecules such as costimulatory factors (CD2, CD28, B7) can be presented to enhance cell-cell adhesion or transmission of additional cell activation signals.

  Another aspect of the invention provides nnAPCs that present peptides associated with allergy and / or autoimmune diseases. For example, a peptide related or derived from IgE is presented with a peptide related or derived from an allergen, or even in combination with a CD40L peptide.

Another aspect of the present invention provides a method for producing a non-naturally occurring antigen-presenting cell (nnAPC) capable of simultaneously displaying up to 10 different peptide molecules associated with allergies and / or autoimmune diseases, The method includes:
a. An insect cell line derived from Drosophila melanogaster eggs is prepared; alternatively, an insect cell line is prepared, where the cells are grown for 12 days, preferably a peptide capable of identifying the desired cell, preferably four Select by mer and then expand the desired cells with OKT3 and IL-2;
b. Growing the insect cells in a medium suitable for growing insect cells, preferably Schneider ™ Drosophila medium;
c. A pRmHa-3 plasmid is prepared from a pRmHa-1 expression vector, wherein the pRmHa-3 plasmid contains an alcohol dehydrogenase gene with a metallothionein promoter, a metal-responsive consensus sequence and a polyadenylation signal isolated from Drosophila melanogaster. Including;
d. DNA complementary to human class I HLA A2.1, B7.1, B7.2, ICAM-1, β-2 microglobulin and LFA-3 was inserted into the pRmHa-3 plasmid, where A2.1 Can be replaced with human class I DNA sequences;
e. The insect cells are transfected with a phshneo plasmid, and the pRmHa-3 plasmid contains complementary DNA;
f. Contacting the insect cell with CuSO ₄ to induce expression of the transfected gene in the insect cell;
Comprising the steps.

Professional antigen-presenting cells such as dendritic cells and macrophages can carry IgE peptides ( Dalvot-Herman et al. (2000) ibid) or IgE recombinant proteins ( Paglia et al. (1996) ibid). Or transduced with a virus encoding IgE or an IgE fragment ( Yang et al., Cellular Immunology (2000) 204: 29-37). These modified professional antigen-presenting cells can then be used to activate IgE-specific CD8 T cells and produce IgE-specific CTLs in vitro. Alternatively, non-professional antigen presenting cells can be transfected or transduced with genes encoding IgE and fragments of IgE in addition to a number of genes encoding a number of costimulatory molecules. Modified non-professional antigen presenting cells can thus be used to stimulate IgE-specific CD8 T cells to generate IgE-specific CTLs.

The insect cells of the present invention are grown in a medium suitable for growing insects (hereinafter referred to as “insect growth medium”). Insect growth media such as Schneider ™ Drosophila media, Grace insect media and TC-100 insect media are commercially available from a number of companies. Alternatively, insect growth media can be prepared by one skilled in the art. Typically, the medium includes inorganic salts (eg, calcium chloride, magnesium sulfate, potassium chloride, potassium phosphate, sodium bicarbonate, sodium chloride and sodium phosphate), various carbohydrates, and chemicals (Imogene Schneider, Exp. Zool . (1964) 156 (1): 91-104), which contains ingredients necessary to promote and maintain insect growth. Alternatively, the medium may contain vitamins, minerals and other ingredients that help the insect ingredients.

  The following is a list of abbreviations and definitions used in this specification.

Abbreviation
APC antigen-presenting cells
CD8 ⁺ CD8 ⁺ T cells
CTL cytotoxic T lymphocytes
Epitope on T cells also known as FAS CD95
ICAM intercellular adhesion molecule
IL interleukin
LFA lymphocyte functional antigen
MHC major histocompatibility complex
nnAPC Antigen-presenting cells that do not exist in nature
PBMC Peripheral blood mononuclear cells
PBS phosphate buffered saline
PCR polymerase chain reaction
RPMI Rosewell Park Memorial Institute (Roswell Park Memorial Institute)
RWJPRI The R.P. W. Johnson Pharmaceutical Research Institute
T target
TCR T cell antigen receptors The following is a list of abbreviations used herein for various epitopes. Individual amino acid residues are readily known to those skilled in the art and are identified according to the single letter code used.
Amino acid abbreviations
3-letter 1-letter alanine ala A
Valine val V
Leucine leu L
Isoleucine ile I
Proline pro P
Phenylalanine phe F
Tryptophan tyr W
Methionine met M
Glycine gly G
Serine ser S
Threonine thr T
Cysteine cys C
Tyrosine tyr Y
Asparagine asn N
Glutamine gln Q
Aspartic acid asp D
Glutamic acid glu E
Lysine lys K
Arginine arg R
Histidine his H

Peptide epitope abbreviation The term IgE 11 as used herein refers to the amino acid sequence of KPCKGTASM (SEQ ID NO: 1).

  The term IgE 209 as used herein refers to the amino acid sequence of IPPSPLDLY (SEQ ID NO: 2).

  As used herein, the term IgE 366 refers to the amino acid sequence of GSNQGFFIF (SEQ ID NO: 3).

  As used herein, the term IgE 29 refers to the amino acid sequence of FPNPVTVTW (SEQ ID NO: 4).

  As used herein, the term IgE 105 refers to the amino acid sequence of HSSCDPNAF (SEQ ID NO: 5).

  As used herein, the term IgE 114 refers to the amino acid sequence HSTIQLYCF (SEQ ID NO: 6).

  As used herein, the term IgE 363 refers to the amino acid sequence of KSNGSNQGF (SEQ ID NO: 7).

  As used herein, the term IgE 307 refers to the amino acid sequence of RSAPPEVYVF (SEQ ID NO: 8).

  As used herein, the term IgE 44 refers to the amino acid sequence of MSTV NFPAL (SEQ ID NO: 9).

  As used herein, the term IgE 411 refers to the amino acid sequence of TSLGNTSLR (SEQ ID NO: 10).

  As used herein, the term IgE 16 refers to the amino acid sequence of TASMTGLGCL (SEQ ID NO: 11).

  As used herein, the term IgE 159 refers to the amino acid sequence of ASTCSKLNI (SEQ ID NO: 12).

  As used herein, the term IgE 125 refers to the amino acid sequence of GHILNDVSV (SEQ ID NO: 13).

  As used herein, the term CD40L 17 refers to the amino acid sequence of LPASMKIFM (SEQ ID NO: 15).

  As used herein, the term CD40L 186 refers to the amino acid sequence of RPFIVGLWL (SEQ ID NO: 16).

  As used herein, the term CD40L 118 refers to the amino acid sequence of DPQIAAHVV (SEQ ID NO: 17).

  As used herein, the term CD40L 220 refers to the amino acid sequence of QSVHLGGVF (SEQ ID NO: 18).

  As used herein, the term CD40L 9 refers to the amino acid sequence of SPRSVATGL (SEQ ID NO: 19).

  The term CD40L 195 as used herein refers to the amino acid sequence of KPSIGSERI (SEQ ID NO: 20).

  As used herein, the term CD40L 252 refers to the amino acid sequence of FSSFGLLLKL (SEQ ID NO: 21).

  As used herein, the term CD40L 7 refers to the amino acid sequence QPSPRSVAT (SEQ ID NO: 22).

  As used herein, the term CD40L 181 refers to the amino acid sequence of EPSSQRPFI (SEQ ID NO: 23).

  As used herein, the term CD40L 79 refers to the amino acid sequence of LSLLNCEMEM (SEQ ID NO: 24).

  As used herein, the term CD40L 152 refers to the amino acid sequence of VMLENGKQL (SEQ ID NO: 25).

  As used herein, the term CD40L 146 refers to the amino acid sequence of TMKSNLVML (SEQ ID NO: 26).

  As used herein, the term CD40L 235 refers to the amino acid sequence of SVFVNVTEA (SEQ ID NO: 27).

  As used herein, the term CD40L 38 refers to the amino acid sequence of GSVLFAVYL (SEQ ID NO: 28).

  As used herein, the term CD40L 19 refers to the amino acid sequence of ASMKIFMYL (SEQ ID NO: 29).

  As used herein, the term CD40L 24 refers to the amino acid sequence FMYLLTVFL (SEQ ID NO: 30).

  As used herein, the term CD40L 167 refers to the amino acid sequence of GLYYIYAQV (SEQ ID NO: 31).

  As used herein, the term CD40L 22 refers to the amino acid sequence of KIFMYLLTV (SEQ ID NO: 32).

  As used herein, the term CD40L 36 refers to the amino acid sequence of MIGSALFAV (SEQ ID NO: 33).

  As used herein, the term CD40L 58 refers to the amino acid sequence of NLHEDFVFM (SEQ ID NO: 34).

  As used herein, the term CD40L 170 refers to the amino acid sequence of YIYAQVTFC (SEQ ID NO: 35).

  As used herein, the term CD40L 26 refers to the amino acid sequence of YLLTVFLIT (SEQ ID NO: 36).

  As used herein, the term CD40L 231 refers to the amino acid sequence of LQPGASVFV (SEQ ID NO: 37).

  As used herein, the term CD40L 45 refers to the amino acid sequence of YLHRRLLDKI (SEQ ID NO: 38).

  As used herein, the term CD40L 147 refers to the amino acid sequence of TMSNNNLVTL (SEQ ID NO: 39).

  As used herein, the term CD40L 229 refers to the amino acid sequence of FELQPGASV (SEQ ID NO: 40).

  As used herein, the term CD40L 160 refers to the amino acid sequence of QLTVKRQGL (SEQ ID NO: 41).

  As used herein, the term CD40L 35 refers to the amino acid sequence of QMIGSALFA (SEQ ID NO: 42).

  As used herein, the term CD40L 185 refers to the amino acid sequence of SQAPFIASL (SEQ ID NO: 43).

  As used herein, the term CD40L 19 refers to the amino acid sequence of ISMKIFMYL (SEQ ID NO: 44).

  As used herein, the term CD40L 153 refers to the amino acid sequence of VTLENGKQL (SEQ ID NO: 45).

  As used herein, the term CD40L 126 refers to the amino acid sequence of VISASSKT (SEQ ID NO: 46).

  The term CD40L 227 as used herein refers to the amino acid sequence of GVFELQPGA (SEQ ID NO: 47).

  As used herein, the term CD40L 20 refers to the amino acid sequence of SMKIFMYLL (SEQ ID NO: 48).

  As used herein, the term CD40L 165 refers to the amino acid sequence of RQGLYYTIYA (SEQ ID NO: 49).

  As used herein, the term IgE 47 refers to the amino acid sequence of SLNGTTTMTL (SEQ ID NO: 50).

  As used herein, the term IgE 96 refers to the amino acid sequence of WVDNKTFSV (SEQ ID NO: 51).

  As used herein, the term IgE 185 refers to the amino acid sequence of WLSDRTTYTC (SEQ ID NO: 52).

  As used herein, the term IgE 309 refers to the amino acid sequence of ALSDRTYTC (SEQ ID NO: 53).

  As used herein, the term IgE 876 refers to the amino acid sequence of SLLTVSGAWA (SEQ ID NO: 54).

  As used herein, the term IgE 883 refers to the amino acid sequence of WLEDGQVMDV (SEQ ID NO: 55).

  As used herein, the term IgE 884 refers to the amino acid sequence of TLTVTSTLPV (SEQ ID NO: 56).

  As used herein, the term IgE 887 refers to the amino acid sequence of QMFTCRVAHT (SEQ ID NO: 57).

  As used herein, the term IgE 890 refers to the amino acid sequence of YATISLLTV (SEQ ID NO: 58).

  As used herein, the term IgE 895 refers to the amino acid sequence of TLCLIQNFM (SEQ ID NO: 59).

As used herein, the term IgE 898 refers to the amino acid sequence of QVMDVDLSTA (SEQ ID NO: 60).
Terms and Definitions As used herein, the term “adoptive immunotherapy” refers to the administration of a donor or autologous T lymphocyte to treat a disease or disease state, where the disease or disease state is a class I HLA molecule. Results in an inadequate or immature immune response normally associated with Adoptive immunotherapy is an appropriate treatment for diseases or disease states where elimination of infected or transformed cells has been shown to be activated by CTLs. For example, but not limited to a disease or disease state, such as melanoma, prostate, breast, colon-rectal, stomach, bronchi and neck, pancreas, neck, ovary, bone, leukemia and lung cancer and / or Tumors; viral infections such as hepatitis B, hepatitis C, human immunodeficiency virus; and malaria; bacterial infections such as Mycobacterium tuberculosis and Listeria monocytogenes.

  As used herein, the term “B7.1” refers to a co-stimulatory molecule that binds to antigen-presenting cells.

  The term “BCNU” as used herein refers to carmustine, also known as 1,3-bis (2 chloroethyl) -1-nitrosourea.

  As used herein, the term “BSE” refers to bovine spongiform encephalopathy.

  As used herein, the term “CD” refers to T lymphocytes (originally), B lymphocytes, monocytes, macrophages and granules grouped by cluster of differentiation, antigenic epitopes and function. Refers to a sphere.

  The term “DTIC” as used herein refers to dacarbazine, 5- (3,3-dimethyl-1-triazeno) -imidazole-4-carboxamide.

  As used herein, the term “ex vivo” or “ex vivo treatment” refers to biological material, typically cells obtained from a patient, or from a suitable alternative source such as a suitable donor. Refers to that the modified and modified cells can be used to treat pathological conditions that are ameliorated by long-term or constant delivery of the therapeutic benefit produced by the modified cells. Treatment includes reintroduction of the modified biological material obtained from either the patient or an alternative source into the patient. The benefit of ex vivo treatment is the ability to provide treatment benefits to the patient without exposing the patient to undesirable collateral effects from the treatment. For example, cytokines are often administered to patients with cancer or virus infection to stimulate the expansion of CTLs in the patient. However, cytokines often cause flu-like symptoms in patients. In the ex vivo procedure, cytokines are used outside the patient's body to stimulate the expansion of CTLs, and the patient is free from cytokine exposure and consequently its side effects. Alternatively, an individual can be treated with a low dose of alpha-interferon at the same time, if appropriate and the individual can benefit from the appropriate circumstances or conditions.

  As used herein, the term “HEPES” refers to N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid buffer.

  As used herein, the term “HLA-A2.1” refers to an HLA class I molecule found in about 45% of Caucasians.

  The term “MPC-10” as used herein refers to a magnetic particle concentrator.

  The term “NK cell” as used herein refers to a natural killer cell.

  As used herein, the term “OKT3” refers to ORTHOCLONE OKT3, muromonab-CD3, anti-CD3 monoclonal antibody.

  As used herein, the term “TAP-1, 2” refers to a transporter associated with antigen processing-1, 2 (Transporter Associated with Antigen Processing-1, 2).

As used herein, the term “Th cell” refers to a helper T cell, CD4 ⁺ .

  As used herein, the term “C-lectin” refers to a peptide of a sequence found to be associated with ovarian cancer.

  As used herein, the term “major histocompatibility complex” or “MHC” is a generic name meant to encompass histocompatibility antigen systems described in different species including human leukocyte antigen (HLA).

  As used herein, the terms “epitope”, “peptide epitope”, “antigenic peptide” and “immunogenic peptide” refer to a peptide derived from an antigen capable of producing a cellular immune response in a mammal. Such peptides can be reactive to antibodies derived from animals immunized with the peptide. Such peptides can be about 5-20 amino acids long, preferably about 8-15 amino acids long, and most preferably about 9-10 amino acids long.

  As used herein, the term “homolog” refers to a functional aspect of the invention described herein, wherein one or more residues are conservatively substituted with a functionally similar residue. Any polypeptide having a sequence of amino acid residues substantially identical to the polypeptide sequence of the present invention is included. Examples of conservative substitutions include substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine with another residue, between arginine and lysine, between glutamine and asparagine, and glycine. Substitution of one polar (hydrophilic) residue such as between serines with another residue, substitution of a basic residue such as lysine, arginine or histidine, or aspartic acid or glutamic acid or Includes substitution of one acidic residue as others.

  As used herein, the term “conservative substitution” includes the use of chemically derivatized residues instead of non-derivatized residues.

  The term “chemical derivative” as used herein refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Examples of such derivatized molecules are, for example, where a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. Including molecules that Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzyl histidine. Also included as chemical derivatives are proteins or peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, proline may be replaced with 4-hydroxyproline; lysine may be replaced with 5-hydroxylysine; histidine may be replaced with 3-methylhistidine; serine may be replaced with homoserine; and lysine with ornithine It may be replaced with. As long as the required activity is maintained, one or more additions and / or deletions of the protein or polypeptide of the present invention or the sequence of the encoded polypeptide will remain with respect to the sequence of the polypeptide corresponding to the nucleic acid sequence of the present invention Any polypeptide having a group is included.

Cytolytic T cells (CD8 ⁺ ) are the main route of defense against viral infection. CD8 ⁺ lymphocytes specifically recognize and kill host cells infected with the virus. Theoretically, it should be possible to utilize the immune system to overcome other types of diseases, including cancer. However, few in vitro / ex vivo procedures have been available to specifically activate CTLs. The identification of important allergic and / or autoimmune antigens noted herein and the specific in vitro activation methods of CTLs described below can now test the concept of adoptive immunotherapy for allergy and / or autoimmune diseases Like that.

All natural T cells require two signals for activation to induce an immune response. For CD8 ⁺ lymphocytes (CTLs), the first signal conveying specificity is the immunogenic peptide fragment (epitope) of the antigen bound to the class I MHC (HLA) complex present on the surface of antigen presenting cells (APCs). Consisting of presentation on CD8 ⁺ lymphocytes. This complex is specifically recognized by the T cell antigen receptor (TCR) that communicates the signal into the cell.

Binding to the T cell receptor is necessary but not sufficient to induce T cell activation and usually does not lead to cell proliferation or cytokine secretion. Full activation requires a second co-stimulatory signal (s) and these signals serve to further enhance the activation cascade. Among the co-stimulatory molecules for antigen-presenting cells, cell adhesion molecules (integrins) such as B7 and ICAM-1 assist this process by binding to CD28 and LFA-1 on T cells, respectively. When a CD8 ⁺ cell interacts with an antigen presenting cell with an immunogenic peptide (epitope) bound by a class I MHC molecule in the presence of an interaction with an appropriate costimulatory molecule, the CD8 ⁺ cell is completely Become cytotoxic T cells.

Lymphocyte-mediated cell killing involves a series of biological events beginning with CD8 ⁺ CTL binding to antigen-targeted (tumor) cells by means recognized by the process described above for T cell activation. Involved. This interaction begins with the binding of antigen bound to APC or MHC class I molecules on target cells to the T cell antigen receptor (TCR). Accessory molecules such as lymphocyte functional antigens (LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1 (ICAM-1), T cell-costimulatory factors (CD2, CD28, B7) -Enhance cell adhesion or transmission of further cell activation signals.

  After cell-cell interactions, CTL kill target cells through the action of soluble cytolytic mediators (perforin and granzyme stored in cytoplasmic granules in T cells) and CTL surface molecules (FAS ligand). After cytolytic attack, target cells die by necrosis (membrane perforation and organelle destruction) or apoptosis (chromatin condensation, DNA fragmentation and membrane foaming).

  The mechanism of lysis mediated by lymphocytes is shown graphically in FIG. In panel A of FIG. 2, after binding to the target cells, the cytoplasmic granules in the CTL are rapidly directed to the target cells in order to release the granules containing perforin and granzyme into the intercellular space. These proteolytic enzymes form pores in the plasma membrane of the target cell and ultimately lead to cell necrosis. In panel B, FAS expression levels on CTL increase after binding to target cells. Interaction of FAS with the FAS receptor on the target cell leads to apoptosis. Enzymes related to proteases such as CPP32 and other IL-1b-converting enzymes (ICE) have been implicated in inducing apoptosis.

Naturally occurring antigen-presenting cells such as dendritic cells, macrophages, autologous tumor cells can be used for in vitro CD8 ⁺ activation. However, the efficiency of activation after this approach is low. This is because natural APC class I molecules contain many types of peptide epitopes in addition to tumor epitopes. Most peptides are derived from normal, harmless cellular proteins and dilute the number of active natural APCs that are actually effective against tumors ( Allison et al., Curr . Op . Immunol . (1995) 7 : 682-686).

A more direct and efficient approach to this problem is to specifically activate CD8 ⁺ cells only with epitopes associated with overcoming specific diseases (such as allergies and / or autoimmune diseases). . For this purpose, artificial antigen-presenting cells are generated by expressing MHC class I molecules in Drosophila melanogaster cells. Since Drosophila (Drosophila) has no immune system, there is no TAP-1, 2 peptide transporter involved to put the peptide epitopes to class I molecules. As a result, the class I molecules appear on the Drosophila cells (Drosophila) as empty conduit. Incubation of these transfected Drosophila cells with exogenous peptides that bind to class I molecules such as, but not limited to, cancer or tumor specific epitopes, including melanoma specific epitopes By doing so, it is possible to occupy each class I molecule with several peptides. High-density expression of class I molecules containing a single peptide in the APC of Drosophila (Drosophila), it is possible to generate a completely specific cytotoxic CD8 ⁺ T cells in vitro antigenic peptide. The method and procedure for preparing Drosophila cells is described in “Cytotoxic T-cells using insect cells expressing human class IMHC and β2-microglobulin, effective June 25, 1996. U.S. Pat. No. 5,529,921, entitled “In Vitro Activation of Cytotoxic T-Cells Using Insect Cells Expressing Human Class I MHC and β2-Microglobulin”, and May 24, 1994 Drosophila cell lines capable of expressing genes encoding MHC class I antigens and β2-microglobulin and assembling empty complexes and the cells Is taught in the preparation method (Drosophila Cell Line Expressing Genes Encoding MHC Class I Antigen And β2-Microglobulin and Capable of Assembling Empty Complexes and Methods of Making Said Cell Lines) title of US Pat. No. 5,314,813 that " ing. In particular, US Pat. No. 5,529,921 discloses various methods for separating and / or enriching precursor cell cultures from column 26, line 56 to column 28, line 22. .

  This feature further eliminates the need for in vivo stimulation of the immune system with various cytokines. This provides a treatment ahead of the side effects caused by cytokines. Alternatively, an individual can be treated simultaneously with a low dose of alpha interferon, if appropriate and under the appropriate circumstances or conditions, the individual can benefit.

  Eliminating the need for in vivo stimulation with cytokines provides improved patient care. Treatment regimes that involve administering cytokines to patients often produce flu-like symptoms such as nausea, nausea and fever. These side effects are generally not life threatening, but can cause life-threatening conditions if a particularly severe reaction occurs in an already weak patient. Another consideration is that such side effects adversely affect patient acceptance and compliance of otherwise beneficial therapies. It provides a treatment regimen that improves patient comfort by eliminating the need for in vivo stimulation with cytokines, and provides an effective treatment that will allow patients to be followed by physicians.

Mouse application of this method, using cells of the T cell receptor (TCR) Drosophila transfected such as APCs and CD8 ⁺ cells derived from the 2C system of transgenic mice (Drosophila) for adoptive immunotherapy Proved in In this system, purified CD8 ⁺ 2C cells are highly capable of in vitro peptides presented by Drosophila cells transfected with MHC class I (L ^d ), which also has co-stimulatory molecules B7-1 and ICAM-1. It is reactive. Drosophila cells were transfected as probes to determine minimal requirements for stimulating unprimed CD8 + T cells ( Cai et al., PNA SUSA . (1996). 93: 14736-14741). Alternatively, the need for co-stimulatory molecules does not apply when using unseparated mouse spleen cells as reactants instead of purified 2C cells. In this case, CD8 ⁺ cells in the spleen group receive “bystander” co-stimulation from activated B cells. Taking advantage of this finding, Drosophila cells transfected with MHC class I (L ^d ) can synergize with normal DBA / 2 mouse spleen cells in vitro without the addition of lymphokines. It was possible to show that responses to specific peptides can be induced. Injection of these CTLs into DBA / 2 mice with P815 mastocytoma led to rapid tumor regression ( Sun et al., Immunity (1996) 4: 555-564).

  The use of any natural or artificial antigen presenting cell (APC) systems to generate cytotoxic T lymphocytes in vitro is limited by the specificity of the antigens these systems can generate. .

The following APC system has been utilized to generate antigen-specific CTLs against a single epitope:
1. Human dendritic cells (DC) that have been pulsed with defined peptides;
2. Peripheral blood mononuclear cells (PBMCs) directed to lymphoblasts and applied with peptides;
3. A lymphoblastoid cell line (LCL) with the natural peptide stripped with acid and loaded with the peptide of interest;
4). Drosophila cells engineered to express empty class I molecules; and mouse 3T3 cells transfected with human class I and co-stimulatory molecules (J-B. Latouche and M. Sadeline, Nature Biotech (2000). ) 18: 405-409).

  Dendritic cells (DCs) are considered primary antigen presenting cell lines in humans due to their broad application in the presentation of primary antigen cells. Self or foreign proteins are processed in DC. The resulting peptide epitope is presented by the HLA molecule and transported to the surface of the DC. However, it has been found that DCs do not consistently generate CTLs directed against four different peptides in vitro. This provided CTLs with activity corresponding to each of the four peptides. It was also found that at the time of peptide application, the DC phenotype was mature or immature and did not affect the results.

Or stimulating Drosophila (Drosophila) usually resulted in CTLs directed to up to 10 different peptides. This provides CTLs that are active against each of the 10 peptides.

The ability of Drosophila cells and DCs to induce CTL responses was determined by the standard stimulation protocol for each of the peptide epitopes, first for each of the peptide epitopes, in order to compare DCs and transfected Drosophila cells. It evaluated according to. Immature DCs were generated by culturing autologous monocytes for 1 week in the presence of IL-4 and GM-CSF. Mature DCs were obtained from immature DCs by adding TNFα to the culture medium 24 hours before harvesting. DCs (immature and mature) are, CD8 cells and peptide - following the procedure used for the application Drosophila (Drosophila) cells stimulated, harvested and applying the peptide, and mixed with purified CD8 cells. Drosophila (Drosophila) cells were generally found to be or irritants than DC. In addition, DCs representing either immature or mature phenotypes were not as efficient as Drosophila cells in inducing specific CTL responses when the defined peptides were used to apply to APC. This is particularly surprising because of the dominant role that DCs play in the immune system.
Preparation of cytotoxic lymphocytes CD8 ⁺ cells isolated from a leukocyte-depleted blood sample by positive selection with anti-CD8 antibody were treated with human class I molecule (HLA-A2.1), B7.1, ICAM-1, Stimulates against IgE and / or CD40L related peptides presented by Drosophila cells expressing LFA-3 and B7.2. CD8 ⁺ cells are restimulated twice with autologous monocytes supplemented with peptide epitopes in the presence of IL-2 and IL-7. CTLs increase non-specifically with OKT3 and IL-2. CTL activity is measured on cells and the purity of CD8 ⁺ T cells is assessed by flow cytometry.

  Manufacturing methods and protocols are performed in accordance with Good Laboratory Practices and Good Manufacturing Practices. “Good laboratory practices” and “good manufacturing practices” are standards for research and manufacturing practices, which are set by the US Food and Drug Administration and are readily known to those skilled in the art. CTLs are monitored for identity, viability, CTL activity, sterility and endotoxin concentration.

  The following examples are provided to illustrate the present invention and are not intended to be limiting.

Production of Drosophila antigen-presenting cells The Schneider S2 cell line was prepared according to published procedures from Drosophila melanogaster (Oregon-R) eggs, and the American Type Culture Collection (CRL 10974). Deposited. S2 cells are grown in commercial Schneider Drosophila (Drosophila) medium supplemented with 10% fetal calf serum.

The pRmHa-3 plasmid vector for expressing MHC class I and costimulatory proteins in S2 cells was derived from the pRmHa-1 expression vector constructed as described in the literature. This includes an alcohol dehydrogenase gene with a metallothionein promoter, a metal responsive consensus sequence and a polyadenylation signal isolated from Drosophila melanogaster .
Complementary DNA for transfection was prepared as follows:
HLA-A2.1 and β-microglobulin: reverse transcription PCR from K562 cells using primers derived from published sequences.
B7.1: Reverse transcription PCR from K562 cells using primers derived from published sequences.
ICAM-1: Reverse transcription PCR from K562 cells using primers derived from published sequences.
B7.2: Reverse transcription PCR from HL-60 cells (ATCC CCL-240) using primers derived from the published sequence.
LFA-3: Reverse transcription PCR from HL-60 cells (ATCC CCL-240) using primers derived from published sequences.

Complementary DNA was individually inserted into the pRmHa-3 vector. S2 cells are HLA-A. 2, B7.1 and a mixture of ICAM-1 plasmid DNA and phshneo plasmid were transfected using calcium phosphate precipitation method. Stably transfected cells were selected by culturing in Schneider's medium containing geneticin. 24 hours prior to use, and the expression of the transfected genes was induced by addition of CuSO _4. Expression levels were assessed by flow cytometry using anti-HLA-A2.1, anti-B7.1 and anti-ICAM-1 antibodies. More than 30% of the cells express HLA is required for efficient in vitro activation of CD8 ⁺ lymphocytes.
Isolated CD8 ⁺ cells of human CD8 ⁺ cells from white blood cells removed blood samples were isolated by positive selection using Dynabead (TM) Isolation method (Dynal (Dynal)). Anti-human CD8 mouse monoclonal antibody (50 μg / ml in human gamma globulin [Gammagard ™]) supplemented with 1% human serum albumin (Baxter-Hyland) and 0.2% Na citrate After incubation for 45 minutes at 4 ° C. with gentle mixing, the cells are washed and coated with sheep anti-mouse IgG coated magnetic beads (Dynabeads ™ (1: 1 bead: cell ratio) Cells and beads are placed in a sterile tube and gently mixed for 45 minutes at 4 ° C. At the end of this time , Cells bound to the antibody are MPC-1 (in accordance with the manufacturer's instructions (Dynal)). Mark) .CD8 cells magnetically retrieved using the separator - the dissociation of the bead complex, CD8 peptide _59-70 (AAEGLDTQRFSG, by incubation for 45 minutes at 37 ° C. in the presence of SEQ ID NO: 52) Free beads are removed magnetically and CD8 cells are counted and analyzed by flow cytometry to assess purity.Recovery of CD8 ⁺ cells is typically greater than 80%. 1 summarizes the cellular composition of 14 separate CD8 ⁺ preparations from normal human PBMC preparations by positive selection with anti-CD8-antibodies.

In vitro immunization of purified human CD8 ⁺ cells
Priming : Drosophila S2 cells are incubated in Schneider's medium supplemented with 10% fetal calf serum and CuSO ₄ (10 ⁶ cells / ml) for 24 hours at 27 ° C. Cells are _harvested , washed and resuspended in Insect X-press medium (BioWhittaker) containing 100 μg / ml human tyrosinase _369-377 (RWJPR) for 3 hours at 27 ° C. After cubation, S2 cells are mixed with CD8 ⁺ cells in RPMI medium (Gibco) supplemented with 10% autologous serum at a ratio of 1:10 and the cell mixture is incubated at 37 ° C. for 4 days. And Drosophila cells die during this time On day 5, IL-2 (20 U / ml) and IL-7 (30 U / ml) are added along with medium change to increase to tyrosinase-specific CTL group .
Re-stimulation : Frozen autologous CD8-deficient PBMCs obtained at the time of leukapheresis were thawed, washed, and 10% autologous serum (as a source of β2 microglobulin) and 20 μg / ml peptide epitope. Resuspend at 10 ⁶ cells / ml in RPMI medium. After γ-irradiation (5,000 rads), the cells are incubated for 2 hours at 37 ° C. Non-adherent cells are removed by washing with Dulbecco's PBS. Adherent monocytes are loaded with tyrosinase epitopes by incubating for 90 minutes in Hepes-buffered RPMI medium containing 10% autologous serum and 10 μg / ml peptide epitope. The supernatant was removed, and Drosophila (Drosophila) - activated CD8 ⁺ cell suspension (in RPMI medium containing 10% autologous serum 3x10 ⁶ cells / ml) 10 CD8 ⁺ cells to one of the adhesive monocytes Add in proportion. After 3-4 days of culture at 37 ° C., IL-2 (20 U / ml) and IL-7 (30 U / ml) are added along with medium change to selectively increase the epitope-specific CTL group.
Non-specific expansion : CD8 non-specifically expanded and in RPMI medium supplemented with autologous serum, anti-CD3 monoclonal antibody (OKT ™ 3), IL-2 and gamma irradiated autologous PBMC Incubate.
Activity and purity assays
CTL assay : Epitope-bearing (target) cells are used as target cells in a ⁵¹ Cr release assay. 5 × 10 ⁶ target cells in RPMI medium containing 4% fetal calf serum, 1% HEPES buffer and 0.25% gentamicin are labeled with 0.1 mCi ⁵¹ Cr for 1 hour at 37 ° C. Cells are washed 4 times and diluted to 10 ⁵ cells / ml in RPMI (HyClone) containing 10% fetal calf serum. In a 96-well microtiter plate, ⁵¹ Cr-labeled target cells loaded with 100 μl effector CTL and 100 μl peptide are combined at ratios of 100: 1, 20: 1 and 4: 1 (effector: target). K562 cells are added at a ratio of 20: 1 (K562) to reduce background lysis of natural killer cells. Non-specific lysis is assessed as described above using cells labeled with ⁵¹ Cr but has no epitope. Controls for measuring spontaneous and maximal release of ⁵¹ Cr are included in duplicate. After 6 hours incubation at 37 ° C., the plate is centrifuged and the supernatant is counted to measure ⁵¹ Cr release.

  The percentage of specific lysis is calculated using the following formula:

Flow cytometry : CD8 ⁺ cells before and after in vitro activation were analyzed for the number of cell surface markers using fluorescent monoclonal antibodies and FACS analysis. The results from a typical activation protocol using cells derived from healthy donors are shown in Table 2.

  In addition to activity and purity, CTL preparations also assay for sterility and endotoxin content.

Trial of cytotoxic T cell injection for IgE producing cells
Test Proposal This example teaches the efficacy of cytotoxic T cell infusion in the treatment of allergic diseases evaluated according to the following factors.
1. Safety and toleration of reinjected autologous CTL after in vitro immunization
2. 2. Dynamics of injected CTL in the systemic circulation considering limiting dilution analysis CTL systemic disposition by radio scintigraphy
4). 4. Cell composition of biopsy nodules by immunohistology (CTL, TH, NK, B cells); Measurable damage regression and response period of 2 months
Treatment with ex vivo generated autologous CTL All patients receive at least one autologous CTL infusion. The number of cycles and cell doses administered to each patient are summarized in Table 1. The number of cells generated in vitro depends on patient-related factors such as the number of PBMC isolated from the aphaeresis method and the number of CD8 ⁺ T cells present in each PBMC preparation. As all of the cells generated in vitro are reinfused into the donor, the dose administered to each patient will necessarily vary. To standardize doses between patients, the calculated “efficacy” score is recorded for each dose. This value is obtained by multiplying the total number of cells by the lytic activity obtained with the target cell bearing the peptide. Patients enter the second, third or fourth cycle of treatment based on their clinical status at the end of each cycle. The total number of natural CD8 ⁺ T cells isolated depends on its proportion in each PBMC preparation. The proportion of CD8 ⁺ T cells varies between patients. The procedure for generating CTL ex vivo is taught in the specification and Example 1 above.
Up-regulation of class I and melanoma-associated antigens in response to IFNα-2b In an attempt to enhance the ability of antigen-specific CTL to lyse IgE-producing cells in vivo, a low dose of IFNα-2b was administered for 5 days prior to CTL infusion. Sequential administration and 3 times a week for an additional 4 weeks. One way to measure the in vivo response to cytokines is to assess the biopsy obtained at successive time points by immunohistochemical analysis for positive staining with specific antibodies.
Antigen Specificity of CTL Generated Ex vivo CTL generated from all patients can be used to produce a T2 target loaded peptide on the day of release, HLA-A2 IgE production M-14, if biopsy material is available to establish the system. Evaluate against clone 4 cell line and autologous M-14 cell line. Each prepared dose of cells is evaluated for its cytolytic activity. T2 cells loaded with either each peptide alone, or a peptide that presents all peptides simultaneously, are used to determine the specificity of the CTL response generated for each patient. The ability to lyse endogenously expressed cells with antigens bound to HLA-A2 is assessed using a system suitable for HLA-A2 or an autologous cell line. In addition to cytolytic activity, antigen-specificity is assessed using established methods to detect intracellular gamma interferon production made in response to specific peptide stimuli. At the end of the ex vivo protocol, the generated CTL are evaluated by this method. Record the percentage of cells specific for each peptide. The total number of specific cells for each large amount of CD8 culture derived from the patient is calculated by adding each peptide specificity detected in that T cell population. An increase in the total number of specific cells is detected with each successive treatment cycle.
The presence of an anergic condition does not exclude the ability to generate CTLs or most patients treated under this protocol that did not prevent clinical response have received prior medical intervention. A pre-treatment skin test is performed to determine if an anergic response to a panel of seven common antigens is not able to generate CTLs ex vivo or correlates with prevention of a proven clinical response. The ability to generate CTL ex vivo does not correlate with the results of the patient's pretreatment skin test.

  IgE plays an essential role in the pathogenesis of allergic asthma. Here we are natural CD8T in which cytotoxic T lymphocytes (CTL) specific for antigenic peptides derived from IgE molecules are resting with IgE peptides presented by artificial antigen-presenting cells. It shows that it can be generated in vitro by stimulating cells. IgE-specific CTL lyses target cells loaded with IgE peptides in vitro and suppresses antigens specific for IgE responses in vivo. Furthermore, adoptive transfer of IgE-specific CTLs into an asthmatic mouse model can suppress the development of lung inflammation and airway hypersensitivity. That is, IgE-specific CTL can provide treatment for allergic asthma and other IgE-mediated allergic diseases.

Cytotoxic T lymphocytes are derived from resting natural CD8 T cells. In the presence of antigen and costimulators, resting natural CD8 T cells are activated and can differentiate into armed cytotoxic T cells, which can destroy target cells expressing the antigen. CTL plays an essential role in immunization against viruses and intracellular pathogens by lysis of infected cells and / or through the effects of cytokines produced by CTLs.
Identification of antigenic peptides derived from IgE protein sequences :
Two alleles of mouse IgE (IgE ^a and IgE ^b ) have already been described (PO6336). The parallel arrangement of IgE ^a and IgE ^b amino acid sequences showed that 95% of the amino acid sequences were identical. A 14 amino acid difference is located in the junction region between CH1 and CH2, and another 5 amino acid difference is located in the junction region between CH3 and CH4. For the amino acid sequence of IgE ^{b for} the 9mer peptide sequence containing binding motifs for L ^d and D ^b MHC class I molecules, see http: // bimas. dcrt. nih. Analyzed by using the Bioinformatics & Molecular Analysis Section software available at gov / molbio / hla bind /. This program ranks potential nonapeptides based on the expected dissociation half time for MHC class I molecules. Based on the ranking analysis, five peptides with eight peptide and D ^b binding motifs with L ^d binding motifs were selected for synthesis (Table 1).

The ability of these synthetic peptides to bind to L ^d and D ^b class I molecules was tested in an MHC class I stabilization assay ( Cai et al. (1996) ibid). Antigen - Transport-deficient ^{(Antigen-transporting deficient) (TAP} -) the RMAS cells ^{(H-2 b)} or ^{L d} transfected RMAS ^{(RMAS-L d)} cells were cultured at 27 ° C. in the presence of titrating concentrations of peptide . After overnight culture at 27 ° C., the cells were cultured for an additional 2 hours at 37 ° C. and the surface expression of L ^d and D ^b on the cells was analyzed by flow cytometry. As shown in Table 1, two IgE peptides, IgE11 and IgE366 are bonded strongly to ^{L d,} whereas, IgE114 bind weakly to ^{L d.} Among the five peptides predicted to bind to D ^b, only IgE44 binding strongly to ^{D b,} and the two peptides IgE16 and IgE125 bind weakly to ^{D b.} Interestingly it was expected to bind to ^{L d} at the beginning IgE366 is bound to both ^{L d} and ^{D b.} That is, a total of 6 peptides were identified as binding to either L ^d or D ^b MHC class I molecules.

In Vitro Generation of CTL Specific for IgE Peptides The ability of these IgE peptides to induce CTL responses was evaluated in vitro. As already described, Drosophila cells transfected with B7-1 and ICAM-1 in addition to MHC class I are potent antigen-presenting cells in the activation of natural CD8 T cells resting in vitro ( APC). Natural CD8T resting cells were purified from lymph nodes of mice and Drosophila (Drosophila) cells loaded with peptide transfected with ^{L d} or ^{D b} plus B7-1 and ICAM, cytokines non Cultured in the presence. IL-2 (20 units / ml) was added on day 3 and thereafter every other day. CTL activity on RMAS (K ^b , D ^b ) cells or RMAS-L ^d cells loaded with peptides was measured on day 9. As shown in FIG. 1, CTL induced by IgE44 peptide specifically lyses RMAS cells loaded with IgE44 peptide, and either target cells alone or target cells loaded with other IgE peptides are IgE44 specific CTLs. Was not recognized by.

Specific CTL activity was induced by IgE16 or IgE125 peptide was shown to bind to ^{D b.} IgE366 is originally identified as ^{L d} binding peptide, interestingly, in addition to inducing ^{L d} restricted CTL by IgE366, IgE366 also induce ^{D b} restricted CTL (Figure 2, Panel B). Among the three ^{L d} binding peptides, IgE11 in addition to IgE366 also induce antigen-specific CTL, killing of IgE specific CTL is perforin-dependent, and (diagram is independent of the expression of IFNγ 2, panel C). Furthermore, CTLs induced by IgE peptides are MHC-restricted because the killing of RMAS targets loaded with IgE44 by IgE44-specific CTLs was completely blocked by anti-D ^b mAbs (FIG. 2, panel D). FACS analysis of these CTLs revealed that they were αβTCR positive CD8 ⁺ T cells and expression of NK cell markers (DX5 or NK1.1) was not detected on these cells (data shown )
Inhibition of IgE response by anti-IgE-specific CTL Since CTL induced by IgE peptides specifically kill target cells in vitro, it was considered interesting if these CTLs could suppress IgE responses in vivo. Mice have very low serum IgE and do not spontaneously produce an allergic response. Ovalbumin precipitated with aluminum hydroxide was used to induce antigen-specific IgE responses in mice. As shown in FIG. 3, after two immunizations using OVA plus aluminum hydroxide, both total serum IgE and egg white specific IgE in the immunized mice are high, and the IgE levels are The mice were further elevated after intranasal boosting with OVA.

^a adult CBF1 / J mice were immunized intraperitoneally with ovalbumin (OVA) plus aluminum hydroxide 50μg per day and 14 days. Two weeks after the second immunization, 5 × 10 6 anti-IgE CTL or control CTL or PBS was given three times every other day. Three weeks after the last CTL treatment, mice were boosted 3 times every other day with OVA in the nose. One day after the last boost, bronchoalveolar lavage fluid was collected and lung tissue was collected from each mouse and stained by HE staining. Inflammation of the lungs of each mouse was assessed separately by a pathologist.
^b score: 0 = normal; T = trace; l = moderate; 2 = moderate to moderate; 3 = moderate; 4 = severe
^c BALT = Bronchial associated lymphocyte hyperplasia

In the presence of specific antigen and costimulation, resting CD8 T cells can be activated and differentiate into CTL, which plays an essential role in the anti-viral immune response. Recently, it has been shown that CTLs specific for antigens associated with tumors generated in vitro can be used to treat cancer patients. We now show that antigenic peptides derived from non-tumor autoantigens can induce specific cytotoxic T lymphocytes (CTLs) in vitro. CTL induced by a peptide identified from CD40L, a self-antigen that is transiently expressed on activated CD4 T cells, can kill activated CD4 cells, and this killing is restricted to restricted class I molecules. It can be blocked either by a specific antibody (Ab) or by an Ab that recognizes the CD8 molecule. Furthermore, neither activated CD4T cells generated from CD40L ^{− / −} mice or from 2m ^{− / −} mice were killed by CD40L-specific CTL, and killing of activated CD4T cells by CD40L-specific CTL is antigen-dependent and It is binding to MHC. Importantly, in vitro generated CTL specific for CD40L suppresses CD4-dependent antibody responses of all isotypes in vivo. In contrast, CTL induced by antigenic peptides derived from IgE suppresses the IgE response, and adoptive transfer to NOD mice at a young stage of CD40L-specific CTL delays the onset of diabetes in NOD mice . That is, in vitro generated CTL specific for non-tumor self-antigen expressed on activated CD4 T cells can modulate immunity in vivo.

  Allergic diseases such as hay fever, asthma and systemic anaphylaxis are immune responses to innocuous substances. Prominent features of the disease are CD4 cell activation and IgE overproduction by B cells. Current therapies focus on symptomatic treatment and do not prevent the onset and progression of the disease. Since allergen-activated CD4 cells and IgE-producing B cells play a central role in the pathogenesis of allergies, our method uses autologous CTL to eliminate activated CD4 T cells and IgE-producing B cells, thereby Prevent the onset and progression of the disease. Two molecules, CD40 ligand (CD40L) and IgE were selected as target antigens for CTL therapy. Three antigenic peptides derived from CD40L and two antigenic peptides derived from IgE were identified. CTLs specific for these peptides were generated and the function of these CTLs was evaluated both in vitro and in vivo.

  Three antigenic epitopes derived from CD40L and two epitopes derived from IgE molecules were identified. Synthetic peptides of antigenic epitopes were able to bind to class I molecules and activate resting natural CD8 T cells in vitro.

CTLs were generated by stimulation of CD8T cells with CD40L or IgE peptides presented by Drosophila cells expressing MHC class I, B7-1 and ICAM-1 molecules. The CTL thus generated in vitro specifically killed the target cells carrying the peptide. CD40L-peptide specific CTL killed activated CD4 T cells and its recognition was dependent on the expression of CD40L and MHC class I molecules.

  The function of CD40L-specific CTL was also evaluated in vivo. The antigen-specific antibody response was suppressed by anti-CD40L CTL. The effects of anti-CD40L CTL and anti-IgE CTL on allergy and autoimmune diseases will be investigated in animal models.

CD8 + T cells were purified from PBMC and cultured with Drosophila cells transfected with A2.1, B7.1 and ICAM-1 in the presence of IgE peptide. Statistics showed the ability of IgE peptides to generate specific CTL responses from different donors. ¹ and ² indicate that anti-IgE CTL was generated from 9-mer and 10-mer, respectively.

The amino acid sequences of the IgE ^a SEQ ID NO: 14 and IgE ^b SEQ ID NO: 50 constant regions were juxtaposed with Vector NTI software. Sequence differences between the two alleles are bold or underlined. Panels A, B, C and D: CD8 ⁺ T cells are expressed in lymph nodes (A, B and D) of CBF1 / J mice, or B6, interferon gamma knockout mice (IFNγ ^{− / −} ) or perforin (PF ^{− / −} ) Purified from knockout mice (C). Purified CD8 T cells were cultured with the indicated IgE peptides presented by SC2 cells transfected with D ^b MHC class I, B7-1 (CD80) and ICAM-1 (CD54) molecules. A low dose of recombinant IL-2 (20 units / ml) was added to the culture on day 3 and every other day thereafter. On day 9, CTL activity was measured against ⁵¹ Cr labeled RMAS cells with or without the indicated IgE peptides. In FIG. 2, panel D, anti-D ^b mAb (20 μg / ml) was added at the beginning of the CTL assay. Adult CBF1 / J mice (8-12 weeks) were immunized intraperitoneally with 50 μg ovalbumin (OVA) precipitated with aluminum hydroxide on days 1 and 14, respectively. Serum IgE, IgG1 and IgG2a were measured by ELISA on day 28. Two weeks after the second immunization, mice were treated with OVA 3 times every other day and boosted intranasally. IgE-specific CTL or control CTL (5 × 10 ⁶ ) were given intravenously 1 day after each boost. Serum IgG and IgE were measured again 2 weeks after the last CTL treatment. Panels A, B, C and D: CBF1 / J mice were immunized as in FIG. Two weeks after the second immunization, the anti-IgE CTL two different doses (5x10 ⁶ and 10x10 ⁶⁾ 1 every other day 3 days was given intravenously. Three weeks after CTL treatment, serum IgE and OVA-specific IgE were measured and OVA was treated three times every other day for booster intranasally. After the last treatment, bronchoalveolar lavage fluid (BAL) was collected and total cells in BAL were counted. Eotaxin in BAL was measured by ELISA and eosinophil cells in BAL were distinguished by HE staining. Panels A and B: CBF1 / J mice were immunized with OVA / Alum on days 1 and 14. Two weeks after the second immunization, mice received injections of 3 treatments, PBS, anti-IgE CTL or control CTL (anti-influenza CTL) every other day. Three weeks after the last treatment, the mice were boosted three times every other day with OVA and boosted intranasally. One day after the last treatment with OVA, each mouse's airway responsiveness to methacholine was measured by whole body plethography. Two independent experiments are shown in panels A and B, respectively. Panels A and B: Adult CBF1 / J mice (8-12 weeks) were immunized intraperitoneally with 50 μg ovalbumin (OVA) precipitated with aluminum hydroxide on days 1 and 14, respectively. Two weeks after the second immunization, mice received IgE-specific CTL (5 × 10 ⁶ ) or PBS intravenously. Three weeks after the last treatment, the mice were treated with OVA every other day for 2-3 treatments and boosted intranasally. One day after the last treatment with OVA, BAL was prepared from each mouse and the lungs from each mouse were fixed and stained with HE. Representative HE staining of lung tissue from mice that received PBS (panel A) or mice that received anti-IgECTL (panel B) was shown. Amino acid sequence deduced from cDNA encoding the human IgE constant region. Total RNA was prepared from a U266 cell line producing human IgE. Total RNA was reverse transcribed and amplified by PCR with two oligos encoding 5 'and 3' human IgE constant regions, respectively. The cDNA was cloned into the pcDNA3 vector and sequenced. Drosophila cells transfected with human HLA-A2 class I cDNA were incubated overnight at room temperature with the indicated concentration of peptide or control peptide (H690) and further incubated at 37 ° C. for 2 hours. Cells were washed and stained with anti-HLA-A2 mAb and analyzed by flow cytometry. Average fluorescence intensity is shown on the Y axis and peptide concentration is shown on the X axis. Panels A, B, C and D: CD8T cells were purified from individual donors and transfected with Drosophila Drosophila transfected with HLA-A2, hB7-1, hB7-2, hICAM-1 and hLFA-3 molecules. The cells were cultured in the presence of the indicated peptides. After 6 days of culture, a low dose of hIL-2 was added to the culture and restimulated with autologous adherent cells loaded with the peptide for an additional 7 days. CTLs were then collected and tested by standard chromium release assay on ⁵¹ Cr labeled T2 cells loaded with peptides that showed specific killing activity. The amino acid sequence of human IgE was derived as described in FIG. Antigenic peptides containing 9 amino acids are underlined and antigenic peptides containing 10 amino acids are shown in bold. TAP2-deficient RMA. S cells RMA transfected with (right panel) or ^{L d.} S cells (left panel) were incubated overnight at 28 ° C. with the indicated concentrations of peptides and then incubated at 37 ° C. for 2-4 hours. Cells were harvested and stained with mAbs specific for L ^d (right panel) or D ^b (left panel) and analyzed with FACScan. CD8 ⁺ T cells are B10. Drosophila cells purified from LN of D2 mice and transfected with L ^d , B7-1 and ICAM-1; and CD40L. Incubated in the presence of 186 peptide (left panel) or QL9 peptide (right panel). IL-2 (20 U / ml) was added to the culture on days 3 and 5. On day 7, ⁵¹ Cr-labeled RMAS. We were measured with respect to L ^d target cells. CD8 ⁺ T cells purified from B6 ^mice, D b, the cells of Drosophila (Drosophila) transfected with B7-1 and ICAM-1, IgE. 44 peptide (left panel) or IgE. Incubated in the presence of 366 peptide (right panel). IL-2 (20 U / ml) was added to the culture on days 3 and 5. CTLs were collected on day 7 and measured against ⁵¹ Cr labeled RMAS target cells in the presence of peptides that showed their specific activity. Panels A and B: Purified CD4 or CD8 T cells were activated with anti-CD3 and anti-CD28 bound to the plate for 40 hours (upper panel) or indicated time (lower panel) and indicated mAb. Stained with 1. CD40L-specific CTL was generated as described in FIG. CD4 cells used as targets were purified from wild type, CD40L ^{− / −} or μ2m ^{− / −} mice and activated with anti-CD3 and anti-CD28 for 40 hours. B10. D2 (upper panel) or B6 (lower panel) were immunized with OVA + CFA and treated as indicated with Ab or CTL. Spleen cells were measured 21 days after immunization by ELISA spot for OVA-producing B cells. Panels A, B, C, D and E B10.2 mice were immunized with OVA + CFA on day 1. Anti-CD40L CTL or anti-CD40L Ab was given on days 1, 3, and 5. Serum was collected on day 14 and OVA-specific immunoglobulins were measured by ELISA. Panel A, B and C: CD8 T cells were purified from C57BL / 6 mice, and ^D b, and cells transfected Drosophila (Drosophila) with B7-1 and ICAM-1, IgE. 44 peptide (A), IgE. Cultured in the presence of 366 peptide (B) and IgE / 125 (C). IL-2 (20 units / ml) was added to the culture on days 3 and 5. CTLs were collected on day 7 and ⁵¹ Cr labeled RMAS. In the presence or absence of peptides that showed their specific killing activity. It was measured with respect to L ^d target cells. CD8 T cells purified from C57BL / 6 (B6), perforin knockout mice (pf ^{− / −} ) and IFNγ knockout mice (IFNγ ^{− / −} ) were transfected with Db, B7-1 and ICAM-1 Drosophila. Cells) and IgE. Cultured in the presence of 44 peptides. IL-20 (20 units / ml) was added to the culture on days 3 and 5. CTL were tested on day 7 and their specific killing activity was determined by IgE. 51Cr-labeled RMAS. In the presence or absence of 44 peptides. It was measured with respect to L ^d target cells. In Panel A, CTL activity was measured in the presence or absence of 10 [mu] g / ml anti -D ^b monoclonal antibody. CD19 ⁺ B cells were purified from human PBMC and cultured with IL-4 (100 ng / ml) and anti-CD40 mAb (5 mg / ml). Anti-IgECTLs were generated in the presence of IgE peptides (B) IgE47 and 96, (C) IgE884 and 890 shown as described in FIG. CTL was added to cultures B and C on day 4. On day 6, culture supernatants were collected and human IgE was measured by ELISA. In culture A, no CTL was added, and in culture D, no B cells were added.

[Sequence Listing]

Claims (9)

A method of producing CTLs specific for one or more non-tumor autoantigen T cell epitopes;
a) isolating CD8 + T cells from the individual;
b) adding T cell epitopes to antigen presenting cells (APC's) with class I MHC molecules;
c) culturing CD8 + T cells with APC's for a time sufficient for activation of precursor CD8 + T cells specific for T cell epitopes;
d) increasing activated CD8 + T cells in culture in the presence of components necessary for the proliferation of activated CD8 + T cells; and e) recovering CD8 + T cells from culture.
Said method comprising.   2. The method of claim 1, wherein the T cell epitope is present on an IgE protein.   2. The method of claim 1, wherein the T cell epitope is present on a CD40 ligand protein.   CD8 + T cells that are specifically cytotoxic to the disease-causing target cell, the target cell having one or more non-tumor self-antigen T cell epitopes that bind to class I MHC molecules on its surface, and CD8 + T The CD8 + T cell, wherein the cell is already selectively activated by interaction with a class I MHC molecule that binds to a non-tumor autoantigen T cell epitope.   5. The method of claim 4, wherein the T cell epitope is on an IgE protein.   5. The method of claim 4, wherein the T cell epitope is on a CD40 ligand protein.   A method of treating a disease mediated by said target cell causing disease wherein the target cell has one or more non-tumor autoantigen T cell epitopes that bind to class I MHC molecules on its surface, wherein such treatment comprises: The method of treatment comprising administering to a patient in need a CD8 + T cell that has already been selectively activated by interaction with a class I MHC molecule that binds to a non-tumor autoantigen T cell epitope.   8. The method of claim 7, wherein the T cell epitope is on an IgE protein.   8. The method of claim 7, wherein the T cell epitope is on a CD40 ligand protein.

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